Method of diagnosing and treating epstein barr virus-based myalgic encephalomyelitis chronic fatigue syndrome patients

ABSTRACT

A method of diagnosing a subset of Epstein Barr Virus, Myalgic Encephalomyelitis Chronic Fatigue Syndrome (ME/CFS) patients through a multi-prong clinical/serological analysis is provided wherein Epstein Barr Virus Abortive Lytic Replication (EBV) is determined as the specific causal agent through the use of serum antibodies to EBV encoded dUTPase and serum antibodies to EBV DNA Polymerase as molecular markers. A method of treating patients diagnosed with Epstein Barr Virus Abortive Lytic Replication (EBV), Myalgic Encephalomyelitis Chronic Fatigue Syndrome (ME/CFS) with specific antiviral nucleosides is also provided, to alleviate the condition.

TECHNICAL FIELD

The present invention relates to a method of classifying, diagnosing andtreating a subset of Epstein Barr Virus, Myalgic EncephalomyelitisChronic Fatigue Syndrome (ME/CFS) patients. In particular, the inventionrelates to a protocol for classifying an appropriate subset of patientsthrough a multi-prong clinical/serological analysis, diagnosing patientswith Epstein Barr virus (EBV) as the specific causal agent of chronicfatigue syndrome through the use of elevated serum antibodies to EBVencoded dUTPase and EBV encoded DNA Polymerase as molecular markers andfurther treating diagnosed patients with specific antiviral nucleosideswhich alleviate the condition.

BACKGROUND

Chronic Fatigue Syndrome (CFS), also known as myalgic encephalomyelitis(ME) and post viral fatigue syndrome, is a life altering illnessaffecting women to men in a ratio of 4:1. To date, evidence-basedetiology or treatment has been elusive. CFS manifestations arelife-altering fatigue in ordinary activities, including constellationsof syncope, chest pain, muscle aches, palpitations, sore throat,low-grade fevers, and inability to exercise without a worsening ofsymptoms, cervical lymphadenopathy, cognitive impairment and resultantdepression.

ME/CFS is not rare. The CDC estimates that there are as many as 500,000persons in the United States who have CFS-like symptoms. However, thedisorder remains debilitating, complex and mysterious in origin, naturalhistory, understanding and treatment.

The spontaneous recovery rate for CFS patients is low, for example, 19%.Numerous treatment regimens have been proposed and includeadministration of various agents such as immune stimulators andsteroids, as well as recommending exercise and psychiatric treatment.While they may lead to modest short-term improvement, such treatmentshave proven generally ineffective in the long run. As the underlyingcauses and distinctions among types of CFS patients have not previouslybeen known, both observational and evidence-based trials have beenmisdirected or inappropriately planned.

While progress has been made to segregate certain groups of CFS patientsand provide them with specific antiviral agents to alleviate thecondition, for other CFS patients—namely those found to have herpesvirus plus co-infections—no effective treatment option has beenidentified to date.

Accordingly, given the distinct types of CFS patients, underlyingcausative agents and varying treatment approaches, there exists a needfor a methodology to identify the appropriate subset of myalgicencephalomyelitis chronic fatigue syndrome patients, a serologicalmethod to diagnose this subset and confirm the causative agent involved,so that a specific treatment protocol can be implemented to alleviatethe CFS symptoms in these patients and restore their ability to lead anormal or near-normal life, free from the debilitating effects ofchronic fatigue.

SUMMARY

In one embodiment of the invention, a method of diagnosing anEpstein-Barr virus subset of Myalgic Encephalomyelitis-CFS patients isdisclosed, including the step of Identifying Epstein-Barr virus AbortiveLytic replication in patients with Myalgic Encephalomyelitis-ChronicFatigue Syndrome by determining the presence of EBV encoded dUTPase orEBV encoded DNA Polymerase antibodies.

In another embodiment, a method of diagnosing an Epstein-Barr virussubset of Myalgic Encephalomyelitis-Chronic Fatigue Syndrome patients isdisclosed and includes the steps of: 1) Determining the presence ofencoded EBV Early Antigen, Diffuse; 2) Determining the presence of EBVencoded DNA polymerase; 3) Determining the presence of EBV encodeddUTPase; and 4) Diagnosing a patient with Epstein-Barr Abortive LyticReplication when EBV Early Antigen, Diffuse is found in conjunction withthe presence of EBV encoded DNA polymerase or EBV encoded dUTPase.

In an additional embodiment, a method of diagnosing the causation agentfor a Myalgic Encephalomyelitis-Chronic Fatigue Syndrome patient isdisclosed and includes the step of: Determining the absence ofEpstein-Barr virus Abortive Lytic replication, through the followingsub-steps: 1)Determining the absence of EBV VCA IgM; 2) Determining theabsence of EBV encoded DNA polymerase; and 3) Determining the absence ofEBV encoded dUTPase through assays for serum antibodies to the early EBVnon-structural proteins of the EBV tegument.

In yet another embodiment, a method of diagnosing and treating anEpstein-Barr virus subset of patients with MyalgicEncephalomyelitis-Chronic Fatigue Syndrome is disclosed and includes thesteps of: 1) Identifying Epstein-Barr virus Abortive Lytic replicationby using EBV—encoded dUTPase and EBV—encoded DNA Polymerase as molecularmarkers; and 2) Treating the subset of patients with the administrationof a therapeutically effective amount of at least one antiviral agent.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a table outlining the CFS Energy Index Point Score;

FIG. 2 is a table depicting the clinical results that establish EBVAbortive Lytic replication in six EBV subset ME/CFS patients treatedwith valacyclovir for over 12 months;

FIG. 3 is a table depicting the clinical results that provide EBVAbortive Lytic Replication in twenty Comparison Group patients; and

FIG. 4 is a flow chart depicting the steps associated with diagnosing apatient with ME/CFS.

DETAILED DESCRIPTION

Reference will now be made in detail to embodiments and methods of thepresent invention which constitute the best modes of practicing theinvention presently known to the inventors. However, it is to beunderstood that the disclosed embodiments are merely exemplary of theinvention that may be embodied in various and alternative forms.Therefore, specific details disclosed herein are not to be interpretedas limiting, but merely as a representative basis for any aspect of theinvention and/or as a representative basis for teaching one skilled inthe art to variously employ the present invention.

Except in the examples, or where otherwise expressly indicated, allnumerical quantities in this description indicating amounts of materialor conditions of reaction and/or use are to be understood as modified bythe word “about” in describing the broadest scope of the invention.Practice within the numerical limits stated is generally preferred.Also, unless expressly stated to the contrary, percent (%), “parts of,”and ratio values are by weight; the description of a group or class ofmaterials as suitable or preferred for a given purpose in connectionwith the invention implies that mixtures of any two or more of themembers of the group or class are equally suitable or preferred;description of constituents in chemical terms refers to the constituentsat the time of addition to any combination specified in the description,and does not necessarily preclude chemical interactions among theconstituents of a mixture once mixed; the first definition of an acronymor other abbreviation applies to all subsequent uses herein of the sameabbreviation and applies mutatis mutandis to normal grammaticalvariations of the initially defined abbreviation; and, unless expresslystated to the contrary, measurement of a property is determined by thesame technique as previously or later referenced for the same property.

It is also to be understood that this invention is not limited to thespecific embodiments and methods described below, as specific componentsand/or conditions may, of course, vary. Furthermore, the terminologyused herein is used only for the purpose of describing particularembodiments of the present invention and is not intended to be limitingin any way.

It must also be noted that, as used in the specification and theappended claims, the singular form “a”, “an”, and “the” comprise pluralreferents unless the context clearly indicates otherwise. For example,reference to a component in the singular is intended to comprise aplurality of components.

Throughout this application, where publications are referenced, thedisclosures of these publications in their entireties are herebyincorporated by reference into this application in their entirety tomore fully describe the state of the art to which this inventionpertains.

The term Chronic Fatigue Syndrome (CFS) and Myalgic EncephalomyelitisChronic Fatigue Syndrome (ME/CFS) are used synonymously herein. As usedherein, CFS is defined to be a disorder caused by infection with aCFS-causing agent. “CFS-causing agent” includes CFS-inducing herpesviruses, for example, HCMV, EBV, and/or HHV6. Based on the CFS—causingagent—2 distinct patient classifications have been created. We name thisgroup A. EB, CMV and HHV6. For group A patients the CFS—causing agent isa CFS—inducing herpes virus such as EBV, HCMV and HHV6. For group Bpatients, with specific co-infections, the encoded proteins maycirculate by the blood stream and peripheral area, causing end-organinjury (e.g. heart, muscle, brain, liver, etc.)

The term “diagnosing: encompasses, for example, characterizing a CFSpatient as belonging to a particular predefined subset of CFS causalgroupings;

The term “treatment” refers to the prevention, partial alleviation orcure of the condition or disorder, or at least one symptom of thecondition or disorder.

The term “effective” or “therapeutically effective” means sufficient tocause at least one of a patient's symptoms to decrease in frequencyand/or intensity. To this end, one measure for effectiveness is theEnergy Index point score (EIPS), which monitors the course of recoveryof CFS patients under treatment by observing an increase in the EIPS of1.0 or more units, and/or a decrease in serological indices ofpathogens.

The term “infection” means the invasion of a host organism's body byanother organism or entity, for example, a virus or bacteria. Infectionby a virus may, but does not necessarily, include entry of the virusinto host cells, production of gene products based on the viral nucleicacid, replication of the virus, and/or further spread of the viruswithin the host body, which may or may not induce an immunologicalresponse by the host organism. “Infection” may include the latentpresence of virus, for example, that which is not replicating, and whosegenes are not being expressed; or, more typically, “infection” mayinclude a virus, at least some of whose genes are being transcribed intomRNA, which may be translated into protein gene products.

“Infection” includes abortive infection and/or replication. As usedherein, “abortive” refers to infections characterized by incompleteviral replications, for example, with non-assembly into a completevirion. Abortive infection can include, for example, expression of thevirus genome to produce early (IE), middle (E) or late (L) gene productsincluding, for example, EBV encoded dUTPase and EBV encoded DNApolymerase. In such an example of abortive infection, the gene productsare not assembled into a complete virus. Abortive infection may include,primarily or exclusively, early only, early and middle, or early, middleand late gene products. Abortive herpes virus replication is a proposedpathogenic mechanism of CFS that can be used to diagnose the disease,and to identify patients who are good candidates for antiviral therapy.

The term “primary abortive replication” includes, for example, firstepisode infection with EBV, HCMV and HHV6. The term “primary abortivereplication” and “primary infection” are substantially equivalent terms.

The term “co-infection” includes infection with, for example, Borreliaburgdorferi, Streptococcus pyogenes, Ehrlichia chaffeensis, Babesiamicroti and Mycoplasma pneumoniae.

“Secondary nonviral infectious agent” includes, for example, Borreliaburgdorferi, Streptococcus pyogenes, Ehrlichia chaffeensis, Babesiamicroti and Mycoplasma pneumoniae. Streptococcus pyogenes infection maymanifest itself as Adult Rheumatic Fever. As used herein “secondaryinfectious agent” and “secondary nonviral infectious agent” aresubstantially equivalent terms.

As used herein, “antiviral agent” includes, for example, valacyclovir,valganciclovir, maribavir, famciclovir and foscarnet. However, anyantiviral agent that is effective against a CFS-inducing infection canbe used according to the methods disclosed herein.

The amount of antiviral agent required to constitute a therapeuticallyeffective amount will vary based on a number of factors, including theseverity of the chronic fatigue syndrome; the identity, age, bodyweight, general health, gender, diet and chemical make-up of thepatient; the type and degree of the cellular response to be achieved;the specific agents or composition employed, and its activity; the timeof administration, route of administration, range of excretion of theagent; the duration of the treatment; drugs used in combination orcoincidental with the specific agent; and like factors well-known in themedical arts. It must also take into consideration the therapeuticwindow, that is, the need to adjust and minimize toxic side-effects. Forexample, it is well within the skill of the art to start doses of theagents at levels lower than those required to achieve the desiredtherapeutic effect and to gradually increase the dosages until thedesired effect is achieved.

Myalgia Encephalomyelitis Chronic Fatigue Syndrome (ME/CFS)

CFS was recognized as a public health problem because of recurrent signsand symptoms of this previously unknown life-altering illness. Cardiac,immune dysfunction, cerebral abnormalities have been identified. Theaverage death age of 144 CFS registered by the National CFIDS Foundationwas 39.3 years, and 20.1% died of suicide, and 20.1% of heart failure.

CFS is generally defined as a disorder of uncertain cause that ischaracterized by persistent, profound fatigue, usually accompanied byimpairment and short-term memory or concentration, sore throat, tenderlymph nodes, muscle or joint pain, and headache unrelated to anypreexisting medical condition, that typically has an onset at betweenthe ages of 30-50 of age. Medline Plus Medical Dictionary. CFS is oftencharacterized by abortive replication and an inability on the part ofthe patient to inactivate the CFS-causing agent by inducing, forinstance, inactive herpes virus latency. CFS patients can respond toantiviral therapy, as measured by, for example, a reduction in nucleicacid gene products of one or more CFS-causing agents.

Clinical tests have shown cardiac, immunologic, radiographic and geneticabnormalities in CFS patients. It was hypothesized that CFS was causedby Epstein-Barr virus (EBV), cytomegalovirus (HCMV), and human herpesvirus 6 (HHV6) in single or multiple virus infection. This paradigmaffirms that the herpes viruses, despite maximum efforts from theseimmuno-competent affected patients, continue an incomplete abortivereplication of middle-gene products, usually without achieving completevirus synthesis. Per this hypothesis, CFS patients are believed tocontinue EBV, HCMV and HHV6 herpes virus replication, and do not achievethe viral latency necessary for recovery. As such, it was proposed thatearly and middle herpes virus (EBV, HCMV and HHV6) gene products toabout the fiftieth gene of these complex viruses, containing over 200open-reading frames, are synthesized without achieving complete virusformation. This hypothesis was tested with the nucleosides valacyclovirfor a suspected EBV CFS subset and valganciclovir for suspected HCMV orHHV6 CFS subsets. As set forth in the International Application underPublication No. WO 2009/054957, by Dr. A. Martin Lerner, incorporated byreference herein in its entirety, specific methodologies have beendisclosed to successfully classify and treat patients with EBV, HCMV andHHV6 in single or multiple infection without co-infection, through theadministration of specific antiviral agents or specified treatmentperiods, as assessed by the validated severity of an illness metric, theEnergy Index Point Score (EIPS). Further information pertaining to Dr.Lerner's work in diagnosing and treating CFS through the use ofantiviral agents can be found in U.S. Pat. Nos. 5,872,123, 6,258,818,6,399,622, 6.537,997 and 6,894,056, which are herein incorporated byreference in their entirety.

As depicted in FIG. 1, the Energy Index (EI) point score (Copyright,Lerner AM and Deeter RG, 1999), is a simple and reliable metric that canbe used to easily evaluate the functional capacity of the CFS patient ateach patient-physician visit. The EI is measured on a scale of 1-10.Validation of the EI was done using two methods: 1.) 20 CFS patients and22 healthy adults, matched for sex, age, place, and time; EI, CFS=3.6;EI, healthy adults=9.9, p=<0.0001; and 2.) 55 CFS patients evaluated atthe same time by the EI and fatigue severity score, correlation 0.67,p=0.0066. Improvement to disappearance of CFS symptoms correlates withan increasing EI.

FIG. 1-Energy Index Point Score Metric Energy Expenditure Grade (Kcalper day**) Activity 0 1715 Bed-ridden, up to bathroom only 1 1750 Out ofbed 30-60 minutes a day (sitting in chair is out of bed) 2 1785 Out ofbed, sitting standing, walking 1-2 hours per day 3 1855 Out of bed,sitting standing, walking 2-4 hours per day 4 1925 Out of bed, sittingstanding, walking 4-6 hours per day 5 1995 Preform with difficultysedentary job 40 hours a week, daily naps Recovery 6 2083 Daily naps inbed, may maintain a 40 hour sedentary work week plus light, limitedhousekeeping and/or social activities 7 2205 No naps in bed. Up 7:00a.m. to 9:00p.m. Able to work a sedentary job plus light housekeeping 82240 Full sedentary workweek, no npas, some social activities plus lightexercise 9 2450 Same as 8 above plus exercise approximately ½ to ⅔normal without excessive fatigue, awakens next morning refreshed10 >2500 Normal *The CFS Energy Index point score can also be determinedby questionnaire (US copyright, 1999 Lerner A. M., Deeter, R. J)reproduced with permission **Kcal per day is calculated for a 70 kg CFSpatient

The validated energy index point score generally can be calculated foreach CFS patient every 3 months at physician visits. A CFS patient hasan EI≦5. A CFS patient with an EI of 0 is bedridden; a CFS diagnosis isno longer present at an EI>5. The EI effect size is 0.25, a mediumeffect size is 0.5. A large effect size is >0.8.

While CFS patients classified with EBV, HCMV and HHV6 in single ormultiple infections without co-infection have been diagnosed and treatedsuccessfully, that is not the case to date for the group of EBV subsetCFS patients with co-infections, such as tick-borne Borreliaburgdorferi, Babesia microti, Anaplasma phagocytophila and/or adultrheumatic fever. For this group of patients, their ability to leadfulfilling and productive lives has to-date remained significantlycompromised. This invention addresses this long standing, but unmetneed.

Epstein-Barr Virus

Epstein-Barr virus (EBV), a gamma herpes virus, is one of the causativeagents of the Chronic Fatigue Syndrome. EBV, like other herpes viruses,encodes for several enzymes that are involved in viral DNA replication;all are part of the early antigen (EA) complex. Several EBV-associatedenzymes have been described to date, such as thymidine kinase (TK),Deoxyribonucleotide polymerase (DNA polymerase), deoxyribonuclease(DNASE), deoxyuridine Triphosphate Nucleotidohydrolase (dUTPase), andribonucleotide reductase, as well as uracil-DNA glycosylase.Historically, while these antibodies to EBV-encoded enzymes wereobserved in patients with different EBV-associated diseases, the reasonfor these antibody patterns and the role these proteins might play inthe pathophysiology of disease, separate from their role in virusreplication has been unknown. Some hypotheses were described in anarticle entitled “Stress-associated changes in the steady-stateexpression of latent Epstein-Barr virus: Implications for chronicfatigue syndrome and cancer” in Brain, Behavior and Immunity 10 (2005)90-103, incorporated by reference herein.

Creation of a ME/CFS Diagnostic Panel & the Identification of MolecularMarkers

A ME/CFS diagnostic panel was created utilizing initialFukuda/Carruthers criteria and a systematic review of 142 ME/CFSpatients. Two groups of ME/CFS patients were found. Group A patients hadelevated serum IgG antibody titers to the herpes viruses Epstein-Barrvirus (EBV), cytomegalovirus (HCMV) and Human Herpes virus 6 (HHV6) insingle or multiple virus infection, but no other co-infections. Group Bpatients had similar elevated herpes virus antibody titers, plusserologic evidence of co-infections, tick-borne (“Borrelia burgdorferi”,“Anaplasma phagocytophilia”, “Babesia microti”); “Mycoplasmapneumoniae”; or adult rheumatoid fever. One hundred and six group AME/CFS patients were followed from 2001-2007, and treated with subsetdirected valacyclovir EBV subset or valganciclovir (HCMV, HHV6 subsets).The data included over 7000 patient visits and 35,000 data entries.Seventy-nine (74.5%) of the group A patients recovered to resume normallife (p<0.0001)—the results are unprecedented.

Notwithstanding the advances made, until now an evidence-based simpletest for the diagnosis of ME/CFS has remained elusive. Dr. Glaser inconjunction with Dr. Williams and Dr. Lerner, independently suggestedthat ME/CFS is abortive lytic herpes virus replication without DNAemia,antigenemia or IgM antibody to herpes virus structural antigens. DrsGlaser and Williams found that the early EBV protein dUTPase producedboth illness behavior in a murine model, and immunologic abnormalitiesin peripheral blood mononuclear cells in vitro. Similar immunologicdisarray with quantitative changes of intracellular performs andgranzymes were found in ME/CFS patients. Since valacyclovir andvalganciclovir do not inhibit early herpes virus proteins, it wassuggested that new herpes virus host cell recruitment had beeninterrupted in the ME/CFS patients who had recovered their health.Valacyclovir after gastrointestinal absorption has a 200×greateraffinity for EBV thymidine kinase than for the host cellular enzyme.(1)Valganciclovir inhibits HCMV and HV6 DNA polymerases. As such, it wasbelieved that herpes virus early proteins may be critical in theetiology of ME/CFS.

In this regard in patients with HCMV subset ME/CFS, a unique presence ofIgM HCMV early proteins p52 (UL44) and CM2 (UL 44-UL 57) was detected in61 HCMV subset patients, but these serum antibodies were not found in acomparison group of well patients. It was thus reported that elevatedserum antibody titers to EBV (EA,D) in 86 of the 106 (81%) ME/CFSpatients with group A ME/CFS were found.

EBV early gene proteins dUTPase and DNA polymerase are enzymes involvedin EBV lytic DNA replication. A repetitive presence of positive serumantibodies to EBV encoded gene products dUTPase and DNA polymerase hasbeen found in 6 EBV subset ME/CFS patients. Over a period of 13-16consecutive months from 2003-2007, serum assays from these 6 EBV subsetME/CFS patients tested positive to EBV dUTPase in 25/50 assays (50%) andto EBV DNA polymerase 40/52 assays (76.9%). Comparison group assays forEBV dUTPase and EBV DNA polymerase from 20 control age-sex matchedpersons were negative. The presence of the EBV proteins dUTPase and DNApolymerase in the blood of ME/CFS patients indicate abortive lyticreplication. Accordingly, the presence of serum antibodies to EBV,dUTPase and EBV DNA polymerase are believed to be diagnostic molecularmarkers for the EBV subset of ME/CFS patients.

Testing Methods

ME/CFS patients

6 ME/CFS patients were identified as Group A EBV subset (five patients),and one patient (group B) had co-infection with Borrelia burgdorferi.The single ME/CFS group B patient had a positive Borrelia burgdorferiwestern blot IgM test. A ME/CFS treatment decision tree that developedfrom the systematic review of the 142 ME/CFS patients is provided asFIG. 4.

Comparison Group

Blood samples were taken from unknown persons at a commerciallaboratory. The age and sex of the comparison group were selected to besimilar to ME/CFS patients.

EBV, VCA, IgM, VCA p18 peptide is a defined VCA-specific marker proteinutilized in the ETI-EBV-M reverse assay (DiaSorin, Inc., Stillwater,Minn. USA). It consists of 56 amino acids of the BFRF encoded VCA andcontains immune-dominant epitopes. This ETI-EBV-M reverse kit utilizesthe enzyme-linked immunosorbent assay (ELISA) based on the antibodycapture technique. The absorbance of the solution measured at 450 nm isrelated to the concentration of IgM to EBV VCA present in the reactionsolution.

EBV-IgG Early Antigen (diffuse), EA(D). The ETI-EA-G kit (DiaSorin) forquantitative detection of IgG antibodies to EBV Early Antigen Diffuse(EBV-EA (D) was used. Diluted serum was incubated with recombinant EA(D)peptide bound to the solid surface of a micro titer well. The ETI-EA-Gassay uses an EA(D) 47 KD recombinant polypeptide. The absorbance of thesolution, measured at 450 nm is proportional to the concentration of IgGantibodies to EBV EA(D) present in the reaction solution.

HCMV ELISA testing for CMV IgG and CMV IgM was performed using ELISAkits from DiaSorin. The HCMV IgG kit contains purified HCMV strainAD-169 antigen-coated wells. The HCMV IgM ELISA is a microcapture assaywith wells coated with anti-human IgM antibody to the same strainAD-169. Sera were diluted 1:10 and incubated for one hour at 37° C. Thewells were washed three times in washing buffer and bound HRP label wasdetected with 3,3′ 5.5 tetramethyl benzidine as substrate for 30 minutesin the dark, after which the color reaction was stopped by the additionof stop solution as recommended by the manufacturer's manual. Theabsorbance was measured at 450/650 nm using Biotech reader (BiotechClinical Laboratories, Inc., Farmington Mich., USA).

Neutralization assays (DNA polymerase, DNase, and dUTPase) wereperformed as previously described. Briefly, 5 μl of human serum weremixed with 5 μl of either purified EBV-encoded dUTPase (3-5 units ofenzyme) or an extract from TPA/sodium butyrate induced Raji cells (forEBV-encoded DNA polymerase) for 30 min at room temperature prior toassaying for enzymatic activity. EBV-encoded DNA polymerase and dUTPaseactivity were determined as described previously. Raji cells wereinduced by treatment with TPA and sodium butyrate for 48 hrs. Cells(106-8) were harvested, re-suspended in 1 ml of extraction buffer (50 mmTris-HC1, pH 8.0 2 mM ATP, 0.2 M KCl, 3 mM dithiothreitol, 2 mM MgCl20.2 mM phenylmethylsulfonylfluoride and 10% (v/v) glycerol, lysed bysonication and centrifuged at 14,000×g for 5 min. The resultingsupernatant was employed for the EBV-encoded DNA polymerase assay.Purified EBV-encoded dUTPase was also obtained as previously described.

For positive controls, assays were performed in the absence of humansera that lacked detectable antibodies to the EBV encoded dUTPase andDNA polymerase and negative controls were performed in the absence ofthe enzyme preparation. Units neutralized were obtained as follows:(Ucontrol-Userum). Serum with neutralizing units greater than or equalto two standard deviations from the control are considered “positive”for dUTPase or DNA polymerase neutralizing antibodies.

The following tests were performed by Lab Corps (Dublin, Ohio) on the 6ME/CFS patients (Groups, A 5 patients, Group B, 1 patient) 163600 Lyme,Western Blot and ELISA, serum—IgG and IgM. Method. Antigen—whole-cellproteins were extracted from B. burgdorferi strain B31, resolved bypolyacrylamide gel electrophoresis into individual antigen bands andthen transferred to nitrocellulose strips for blotting.

Babesia microti Antibody Panel—IgG and IgM. Method—IFA. Antigen—thesubstrate for the IFA was guinea pig or hamster erythrocytes infectedwith Babesia microti organisms and then fixed onto microscope slides.Upon interaction with human sera containing anti-Babesia antibodies andthe appropriate conjugate, infected cells fluoresce.

138315 Ehrlichia Ab panel “(Granulocytic and Monocytic/Anaplasmaphagocytophilia)”—IgG and IgM. Method: IFA. Antigen: is eitherinactivated HGE or HME

163758 Mycoplasma pneumoniae Antibodies—IgG and IgM. Method: EIA.Antigen: Mycoplasma pneumoniae FH antigen

006031 Antistreptolysin 0 Ab. Method: Latex immunoturbidimetry. HumanAntistreptolysin 0 antibodies agglutinate with latex particles coatedwith streptolysin 0 antigens. The precipate is determinedturbidimetrically at 552 nm.

Results ME/CFS Patients (FIG. 2)

Demographics. ME/CFS patients (FIG. 2) the six ME/CFS patients (5 women)were selected from 142 ME/CFS of the 2001-2007 systematic review. Theywere 37-59 years old. Serum samples were taken at intervals Mar. 5,2002-Nov. 14, 2003. There were 7 to 10 sera from each patient. Five ofthe 6 ME/CFS patients were Group A (no co-infections), and one patienthad co-infection with Borrelia burgdorferi (patient no. 2). Five of the6 patients were HCMV IgG serum antibody negative. Initial EIPS valueswere 3.5-5.0, meaning that patients could be out of bed only 3 to 4hours a day, and required daily naps in bed to complete each day. OneME/CFS patient was able to struggle to complete a sedentary working day(FIG. 2, patient no. 6). The solitary man of these 6 “ME/CFS” patientsdid not meet criteria for ME/CFS at baseline (EIPS, 6). He struggled atbaseline to maintain his sedentary working day, required a daytime nap,and could no longer do any exercise without marked syncope and worseningfatigue. One year later the final EIPS values were 7-8, for the 5 groupA ME/CFS patients, meaning that patients could now live normal lives.The single group B ME/CFS patient's final EIPS value increased from abaseline of 3.5 to 5, but this woman still met international criteriadiagnosis of ME/CFS. The EIPS is a validated (FSS-9 item scale with highdegree of internal consistency measured by Cronbach's alpha) is aFunctional Activity Appraisal: energy Index Score Healthcare workerAppraisal.

EBV Encoded Gene Products EBV, VCA IgM Forty-nine VCA IgM assays weredone. All were negative.

EBV, EA (D) Forty-nine EA (D) assays were done. All were positive excepttwo sera from ME/CFS patient number 6 whose baseline EIPS value was 6.Mean patient EBV (EA) titers were: 54 (patient one); 123 (patient two);63 (patient three); 128 (patient four); 49 (patient five); and 27(patient six), negative <20. The mean EA (D) titer for these 6 patientswas 74.

EBV dUTPase Three of 9 (33.3%), ME/CFS (patient one); 5 of 7 (71.4%)ME/CFS (patient two); 3 of 9 (33%) ME/CFS (patient three); 8 of 10 (80%)ME/CFS (patient four); 3 of 8 (37.5%) ME/CFS (patient five); 3 of 7(43%) ME/CFS (patient six) were positive assays for elevated serumantibody titers to EBV dUTPase. Twenty-five of the 50 (50%) assays werepositive.

EBV DNA polymerase. Eight of 10 (80%) of ME/CFS (patient one); 4 of 7(57.1%) ME/CFS (patient two); 7 of 10 (70%) ME/CFS (patient three); 9 of10 (90%) ME/CFS (patient four); 7 of 8 (88%) ME/CFS (patient five); and3 of 5 (71.4%) (patient six) were positive serum assays for elevatedantibody titers to EBV DNA polymerase. Forty of the 52 (76.9%) ME/CFSassays from ME/CFS patients were positive.

Comparison Group Patients (FIG. 3) Demographics

The mean age of the comparison group was 48.7 years (36-59). Fifteen of19 (78.9%) persons were women. EBV, VCA, IgM Twenty assays from thecomparison group were done. All were negative.

EBV, EA (D) Twenty assays from the comparison group were done. Fourteencomparison group patients had negative EV EA (D) titers. Six comparisongroup patients had EBV (EA) titers. The mean EA (D) of the comparisongroup was 22.

EBV dUTPase twenty assays from the comparison group were done. All werenegative.

EBV DNA polymerase twenty assays from the comparison group were done.All were negative.

Analysis of FIGS. 2 and 3

These data demonstrate the presence of elevated serum antibodies toencoded EBV non-structural proteins DNA polymerase (EBV poly) anddUTPase (EBV dUTP) in blood from six EBV subset ME/CFS patients. Theseantibodies in the blood samples of ME/CFS patients accompany theirlikely source, a “primary” plasma cell apoptosis. EBV poly anddUTP-emias are being monitored in the ME/CFS patients. It is believedthat EBV lytic virus had likely originated in pharyngeal epithelialcells to then infect adjacent memory B cells where the EBV genome waslatent. As memory B cells differentiate to plasma cells, they arebelieved to be the probable site of the current early encoded EBVproteinemia abortive lytic replication. While the early EBV proteinBZLF1 (Zta, EB1) initiates the EBV viral lytic cycle, only 15% of Bcells expressing BZLF1 achieve a full lytic cycle to primary infection(infectious mononucleosis)—completing the virion. The majorityreplication is abortive lytic in type. It is believed that BZLF1 anddUTPase in vitro induce a cellular dysregulation, with TNF andFasligand. It is further believed that the encoded circulating proteinsBZLF1, dUTPase, DNA polymerase and, likely, other EBV encoded tegumentearly proteins may enter host cells of affected ME/CFS patient's organs,namely heart, striated muscle, and the brain to initiate a secondaryexpansive apoptosis along with IL-6, I-10, TGF-B, tyrosine Kinase, TKTmatrix metalloproteinase and C-Fos. This secondary ME/CFS apoptosis isbelieved to be the pathologic mechanism of ME/CFS and does not requireEBV DNA, and further may de-mystify and explain the difficulty inassociating EBV replication and ME/CFS.

The proteins EBV dUTPase and EBV DNA polymerase separate the EBV subsetME/CFS patients from 20 comparison group patients, and, thus, offer apossible molecular marker for the diagnosis of EBV subset ME/CFSpatients. Assays for EBV, VCA IgM; EBV, EA(D); EBV dUTPase and EBV DNApolymerase along with Fukada/Carruthers ME/CFS criteria and theconfirming diagnostic panel described define the EBV subset of ME/CFSpatients.(FIG. 2) Five of the six ME/CFS patients at baseline metinternational criteria for ME/CFS. The single male was chronically ill,could not work regularly or exercise.

Forty-nine EBV VCA IgM serum assays taken serially over 13-16 monthswere negative. Comparison group assays for EBV VCA IgM, EBV dUTPase andEBV DNA polymerase were negative. However, 47/49 (95.9%) EBV EA (D)assays from ME/CFS patients were positive. Twenty-five of 50 (50%)dUTPase antibody serum assays were positive and 40/52 (76.9%) DNApolymerase serum antibody assays were positive from the ME/CFS patients.The results describe abortive lytic EBV replication of early viralproteins EA (D) dUTPase, and DNA polymerase which have been releasedfrom infected cells into the blood. These aberrant virustegument-belonging, intracellular belonging proteins and, perhaps, otherearly EBV proteins may traverse cellular membranes of multiple hostsystems stimulating the immune dysregulation and the symptoms of ME/CFS.Serum antibodies of EBV dUTPase and EBV DNA polymerase in blood samplesof ME/CFS patients, who have been treated or not treated withvalacyclovir are present for over 400 days.

EBV dUTPase (BLLF3) is a part of this EBV early antigen complex. EBV DNApolymerase (BALF5) is an early protein heralding EB lytic replication.EBV DUTPase catalyzes the hydrolysis of dUTP to dUMP, preventingincorporation of uracil into replicating DNA. EBV DNA polymerase isnecessary for EBV lytic replication. EBV dUTPase and EBV Zta (BZLF1,2EBRA, EB1) induce widespread immune dysregulation. Accordingly,abortive lytic EBV replication may be responsible for thecardiomyopathy/encephalopathy of ME/CFS.

In addition to the EBV subset of ME/CFS patients, space flights alsolead to reduced T-cell function, altered leukocyte and lymphocytesubsets, decreased delayed type hypersensitivity and altered cytokineproduction. Elevated levels of glucocorticoids are present during andafter space flights. B-lymphocytes from 6 astronauts who flew ˜180 dayspace flights showed expanded expression of EBV latent, immediate earlyand early gene transcripts and EBV late replicative transcription uponreturn to earth. Under such conditions, EBV undergoes abortive lyticreplication like the EBV subset ME/CFS patients.

FIG. 2 EBV ABORTIVE LYTIC REPLICATION IN SIX EBV SUBSET ME/CFS PATIENTSTREATED WITH VALACYCLOVIR FOR ≧12 MONTHS Patient EA(D) VCA, IGM DNA No.Age Sex Sera Date (<20) (<20) dUTPase Polymerase 1 37 F 1 Mar. 12, 200253 Negative Positive Negative Group A 2 Apr. 23, 2002 Not Not doneNegative Positive ME/CFS done Co- 3 Jun. 27, 2002 50 Negative PositivePositive infection 4 Sep. 28, 2002 36 Negative Negative Positive None 5Oct. 22, 2002 74 Negative Not done Positive EIPS 5 baseline 6 Nov. 21,2002 115 Negative Negative Positive EIPS 8 7 Jan. 9, 2003 70 NegativeNegative Negative final 8 Mar. 6, 2003 51 Negative Negative Positive 9May 1, 2003 45 Negative Positive Positive 10 Jun. 10, 2003 42 NegativeNegative Positive Totals 10 sera 14 months 54 (mean) 0/9 (0%) 3/9 (33%)8/10 (80%) Patient EA(D) VCA, IGM DNA No. Age Sex Serum Date (<20) (<20)dUTPase Polymerase 2 43 F 1 Mar. 22, 2002 116 Negative Positive PositiveGroup B 2 May 17, 2002 125 Negative Positive Positive ME/CFS 3 Sep. 26,2002 120 Negative Positive Positive Co- 4 Oct. 24, 2002 123 NegativePositive Positive Infection 5 Nov. 21, 2002 150 Negative NegativeNegative Lyme 6 Mar. 12, 2003 104 Negative Negative Negative Disease 7May 9, 2003 121 Negative Positive Negative EIPS 3.5 baseline EIPS 5final Totals 7 sera 13 months 123 (mean) 0/7 (0%) 5/7 (71.4%) 4/7(57.1%) Patient DNA No. Age Sex Sera Date EA(D) VCA, IGM dUTPasePolymerase 3 46 F 1 Mar. 12, 2002 63 Negative Positive Negative Group A2 Apr. 19, 2002 70 Not done Negative Positive ME/CFS 3 Jun. 28, 2002 45Negative Positive Positive Co- 4 Aug. 5, 2002 75 Negative NegativePositive infection 5 Sep. 17, 2002 54 Negative Not done Positive None. 6Oct. 31, 2002 100 Negative Negative Positive EIPS 5 7 Nov. 21, 2002 NotNegative Negative Negative baseline done EIPS 8 8 Feb. 6, 2003 47Negative Negative Positive final 9 Mar. 20, 2003 67 Negative PositivePositive Co- 10 Jun. 20, 2003 49 Negative Negative Positive morbidityCancer of breast Totals 10 sera 14 months 63 (mean) 0/9 (100%) 3/9 (33%)7/10 (70%) Patient DNA No. Age Sex Serum Date EA(D) VCA, IGM dUTPasePolymerase 4 59 F 1 Jun. 19, 2002 150 Negative Positive Positive Group A2 Aug. 6, 2002 122 Negative Positive Negative ME/CFS 3 Oct. 8, 2002 129Negative Positive Positive Co- 4 Nov. 7, 2002 184 Negative NegativePositive infection 5 Dec. 3, 2002 149 Negative Positive Positive None 6Feb. 4, 2003 131 Negative Positive Positive EIPS 4 7 Mar. 18, 2003 134Negative Positive Positive baseline 8 May 15, 2003 86 Negative PositivePositive EIPS 7 9 Jul. 10, 2003 89 Negative Positive Positive final 10Nov. 14, 2003 105 Negative Negative Positive Co- morbidity Cancer ofbreast Totals 10 sera 16 months 128 (mean) 0/10 (0%) 8/10 (80%) 9/10(90%) Patient DNA No. Age Sex Serum Date EA(D) VCA, IGM dUTPasePolymerase 5 59 F 1 Apr. 4, 2002 38 Negative Positive Negative Group A 2May 16, 2002 58 Negative Negative Positive ME/CFS 3 Jun. 27, 2002 Notdone Not done Negative Positive EBV/HCM 4 Aug. 18, 2002 45 NegativeNegative Positive V subset 5 Oct. 29, 2002 43 Negative Negative PositiveEIPS, N/A 6 Dec. 12, 2002 54 Negative Negative Positive Diabetes 7 Apr.10, 2003 75 Negative Positive Positive Mellitus 8 May 30, 2003 27Negative Positive Positive Type 2 Congestive heart Failure Totals 8 sera13 months 49 ( mean) 0/7 (0%) ⅜ (37.5%) ⅞ (88%) Patient DNA No. Age SexSerum Date EA(D) VCA, IGM dUTPase Polymerase 6 59 M 1 Mar. 5, 2002 Neg.Negative Positive Negative Group A 2 Apr. 6, 2002 25 Negative NegativePositive ME/CFS 3 Sep. 11, 2002 32 Negative Positive Negative Co- 4 Dec.19, 2002 23 Negative Positive Positive infections- 5 Mar. 14, 2003 Neg.Negative Negative Positive None 6 May 5, 2003 28 Negative NegativePositive EIPS 6 7 Jun. 17, 2003 Neg. Negative Negative Positive baselineEIPS 7 final Totals 7 sera 14 months 27 (mean) 0/7% (0%) 3/7 (43%) 5/7(71.4%)

FIG. 3-EBV Abortive Lytic Replication in Twenty Comparison GroupPatients Patient EA(D) VCA,IGM DNA No. Age Sex Sera Date (<20) (<20)dUTPase Polymeras  1 38 F 1 Jun. 28, 2011 68 Negative Negative Negative 2 43 F 2 Jun. 28, 2011 8 Negative Negative Negative  3 45 F 3 Jun. 28,2011 75 Negative Negative Negative  4 59 F 4 Jun. 28, 2011 9 NegativeNegative Negative  5 59 F 5 Jun. 28, 2011 24 Negative Negative Negative 6 59 M 6 Jun. 28, 2011 13 Negative Negative Negative  7 37 F 7 Jun. 28,2011 54 Negative Negative Negative  8 44 F 8 Jun. 28, 2011 12 NegativeNegative Negative  9 46 F 9 Jun. 28, 2011 12 Negative Negative Negative10 59 F 10 Jun. 28, 2011 9 Negative Negative Negative 11 59 F 11 Jun.28, 2011 6 Negative Negative Negative 12 60 M 12 Jun. 28, 2011 7Negative Negative Negative 13 59 M 13 Jun. 28, 2011 9 Negative NegativeNegative 14 47 M 14 Jun. 28, 2011 Not Negative Negative Negative done 1515 Jun. 28, 2011 10 Negative Negative Negative 16 43 F 16 Jun. 28, 20119 Negative Negative Negative 17 44 F 17 Jun. 28, 2011 19 NegativeNegative Negative 18 36 F 18 Jun. 28, 2011 35 Negative Negative Negative19 38 F 19 Jun. 28, 2011 6 Negative Negative Negative 20 50 F 20 Jun.28, 2011 53 Negative Negative Negative Comparison (Mean) 15/19 Jun. 28,2011 6/20 > 20 0/20 0/20 0/20 (0%) Group 48.7 yrs 78. (30%) (0%) (0%)positive (36-59) 9% Positive Female 22 (mean) ME patients 57.0 yrs ⅚Jun. 28, 2011 47/49 0/49 25/50 40/52 (37-59) 83. >20 (0%) (50%) (76.9%)3% (95.9%) positive positive positive Female positive 74 (mean)

In another aspect, the invention provides methods of diagnosing an EBVsubset of CFS patients, comprising selecting a set of target serologicmarkers of a plurality of pathogens, wherein the pathogens areassociated with CFS—in this case the identification of the molecularmarkers for EBV, DNA polymerase and dUTPase; obtaining a set ofquantitative values for a reference level for each of the serologicmarkers in the set, wherein a level of serologic marker above thereference level indicates the presence of a pathologic level of thepathogen in the physiological fluid tested; obtaining from the patient asample of physiological fluid in which the target serologic markerswould be found if the pathogen is present in the patient; measuring theserologic levels for each of the target serologic markers in thephysiologic fluid of the patient in obtaining a quantity that value forthe serologic level of each target serologic marker, comparing theserologic level with the reference level for each target serologicmarker; and identifying the patient as having EBV abortive lyticreplication if the identified molecular markers are significantly abovethe reference level. As used herein, a serologic level is significantlyabove the reference level if the serologic level exceeds the referencelevel to the degree that it would indicate the presence of an infectionto a person of ordinary skill in the art. “Serologic marker” encompassesany serologic evidence that indicates the presence of the pathogen inthe patient's body. Such evidence can include, for example, the presenceof a molecule or other entity—such as the two molecular markersidentified herein for the EBV subset of CFS patient—that is generallynot present in the healthy individual; increase or decrease of the levelof the molecule or other entity of what is generally present in healthyindividual; or any other indicator know in the arts.

During the initial primary herpes virus infection, EBV antibodies tomultiple early, middle and late gene products, as well as those tocomplete virus particles, are produced, which may give rise toserum-specific IgM antibody titers two multiple gene products. Then, asthe patient recovers all serum antibody titers to early, middle and lateherpes virus nonstructural genes disappear, any only positive serumantibody titers to complete structural virions, such as serum-specificIgG remain.

In CFS, however, there is “abortive” herpes virus infection with nocompete virion multiplication, and products in the periphery may induceelevated serum antibody titers.

Therefore, in EBV subset CFS there may be elevated serum antibody titersto EBV early, middle and late gene products not ordinarily present. Ingroup A CFS in which abortive replication by two or three of the EBV,HCMV, HHV6 is present, appropriate dual or triple herpes virus elevatedearly, middle, late serum antibody titers to gene products may be found.

Experimental Approaches to Developing Serological Assays

To specifically diagnose ME/CFS, serological assays need to be preparedbased on EBV viral products specific to the ME/CFS diagnosis. As such,in one embodiment, a combination of several gene products specific forviral replication, representing active viral replication will be used todevelop multiplex immunoassays. Such immunoassays will preferably bedeveloped on a Luminex TM 100×MA platform using microsphereimmunofluorescent bead technology capable of simultaneously analyzing asingle specimen for multiple analytes.

Analytical validation are preferably performed for each antigen in asingleplex assay, and then all together in a multiplex assay.Preferably, analytical sensitivity and specificity within the detectionlimit are performed for each antigen individually in a singleplex assayand then combined in a multiplex assay.

a) Selection of Antigens Based on EBV Viral Genes

Candidates for IgM and/or IgG should be assayed and developed. Differentantigens may be used for this assay, such as: 1) For IgG serologicalassay, EBV-EA composed of EBV-EA-D recombinant proteins which arespecific for EBV IgG will be used as an internal control to monitor theIgG response to these antigens; 2) For EBV IgM serology, two newantigens may be used for this assay: EBV encoded dUTPase and the EBV DNAencoded polymerase gene products in combination, and EBV VCA IgM or VCAp18 peptide (considered as IgM for EBV serology) will be used; and 3)EBV VCA IgM will be used as an internal control for IgM serology.

EBV encoded dUTPase and the EBV DNA encoded polymerase gene productshave been shown to be specific for abortive lytic EBV replication inME/CFS patients and thus, developing a multiplex immunoassay using thisspecific combination of highly defined recombinant viral proteins willoffer a unique diagnostic tool for detection and differentiation of EBVinfection in ME/CFS patients. In addition, this test's utility can beexpanded to serological testing for infection in immuno-compromisedpatients including those infected with HIV.

b) Cloning of the Candidate Genes

Plasmids with clones of EBV encoded dUTPase and the EBV DNA encodedpolymerase gene products are available and can be expressed using knowncloning plasmid and expression systems.

c) Production and Purification of Antigens (Cloned Genes)

While antigens will generally be produced in a laboratory, EBV EA andEBV VCA IgM antigens are commercially available.

d) Luminex Technology Platform.

Luminex's xMAP technology, based upon flow cytometry, color-codes tinybeads, called microspheres, into 100 distinct sets. Each bead set can becoated with a reagent specific to a particular bioassay (antigens)allowing the capture and detection of specific analytes from a sample.In this way, xMAP technology is well suited to assay development byallowing multiplexing of up to 100 unique assays within a single sample,both rapidly and precisely.

e) Selection and Activation of Luminex Microsphere Beads

The antigens will be coupled to the Luminex microspheres by usingN-hydroxysulfosuccinimide enhanced carbodiimide-mediate couplingreaction.

f) Covalent Coupling of Selected Antigens to Luminex Microspheres(beads)

In a preferred embodiment, the selected antigens: EBV EA and EBV VCA-IgMand EBV encoded dUTPase and the EBV DNA encoded polymerase geneproducts/antigen will be diluted in a Phosphate Buffering Solution (PBS)to a concentration of 12 μg/ml. A total of 500 μl of theantigens/antibodies (12 μg/ml) will be added to the activatedmicrosphere and vortex on a low setting for 10-20 seconds to re-suspendthe microspheres. The antigens will be coupled to the microsphere asfollows: EBV EA to microsphere #32, EBV VCA IgM to microsphere # 53, EBVencoded dUTPase to microsphere #38, EBV DNA encoded polymerase tomicrosphere #25. Calibrators containing different concentrations of EBVEA and EBV IgM will serve as an internal control that will monitor thevalidity of the assay, they will be covalently attached to microspheres#1, #10, #15 and #20. These selected microspheres are different incolor, which aids spectral resolution. Similarly, the listed antigenswill be individually coupled to selected microspheres.

g) Multiplex Luminex Immunoassay

The Luminex Multiplex Immunoassay is designed to detect specific EBVantibodies in human sera to a variety of EBV antigens. The testprocedure involves two incubation steps:

Step 1. Test sera (properly diluted) are incubated in a vesselcontaining a multiplexed mixture of the bead suspension. The multiplexedbead suspension contains a mixture of distinguishable sets ofpolystyrene microspheres. For IgG; Three of these bead sets areconjugated with the EBV-EA, EBV encoded dUTPase and the EBV DNA encodedpolymerase gene products.

For IgM serology the multiplexed bead suspension contains a mixture ofdistinguishable sets of polystyrene microspheres; Three of these beadsets are conjugated with the EBV-VCA IgM, EBV encoded dUTPase and theEBV DNA encoded polymerase gene products. The bead mix also contains onebead set designed to detect non-specific binding and four separate beadsets are used for assay calibration. If present in patient sera,specific antibodies will bind to the immobilized antigen on one or moreof the bead sets. The microspheres are then rinsed to removenon-reactive serum proteins.

Step 2. Phycoerythrin-conjugated goat anti-human IgG (Fc chain specificfrom Chemicon Inc) is added to the vessel and the plate is incubated.The conjugate will react with IgG antibody immobilized on the solidphase in step 1. The bead suspension is then analyzed by the Luminex 100instrument. The bead set(s) are sorted (identified) and the amount ofreporter molecule (PE conjugate) is determined for each bead set.Internal calibration bead sets are used to convert raw fluorescence intooutcome (units).

h) Optimization and Validation of the Assay (Test Verification)

Analytical validation will be performed for each antigen in a singleplexassay, and then all together in a multiplex assay. Analyticalsensitivity and specificity with limit of detection will be performedfor each antigen individually in a singleplex assay and then alltogether in a multiplex assay. Different concentrations of each antigenwill be used for sensitivity and specificity studies ranging from thelowest detection limit through the highest detection limit. In addition,reproducibility, repeatability and precision studies will be performedusing negative, low positive and high positive controls by testing thesecontrols in multiple repeats within the run, between the run, overdifferent days, different lots and different operators. Mean, standarddeviation and CV will be calculated. Optimization will be performeduntil reproducibility and precision indicates a CV% less than 10.Studies will be compared with the existing EBV ELISA assay performed inthe laboratory (Diasorin, Inc). After the optimization of each antigen,all of the antigens together will be compared with ELISA testing.Internal controls and internal calibrations will be also optimized incomparison to the ELISA assay. In addition other conditions, such asincubation time, wash solution and sample dilution as well as storageconditions will be optimized.

i) Reference Ranges.

Reference ranges will be established through base comparison with ELISAtesting. Negative specimens will be measured by ELISA and then byLuminex multiplex assay to establish reference ranges. In additionpositive patient specimens will be diluted to non-detection levels toconfirm established reference ranges.

j) Result Interpretation

In the IgG serology, EBV EA must be positive and should validate eachreaction/assay. The control attached to bead #1 must be always negative,control attached to bead #10 must be weak negative, control attached tonumber #15 must be positive and control attached to #20 must be stronglypositive. After all controls are positive, antibodies to either of thetwo new antigens: EBV encoded dUTPase and the EBV DNA encoded polymerasegene products must be positive for the test to be positive.

k) Analytical Sensitivity

Analytical sensitivity will be studied in two ways: first, positiveresults will be diluted to the lowest detection levels and compared toELISA assays, and second, pooled negative specimens will be spiked withknown concentration of antibodies to EBV encoded dUTPase and the EBV DNAencoded polymerase antigens and then assayed by both Luminex and ELISA.

l) Analytical Specificity

Analytical specificity will be studied by spiking pooled negativepatient specimens with antibodies to EBV encoded dUTPase and the EBV DNAencoded polymerase gene product.

m) Precision

For precision six specimens will be tested. On each day of testing, eachsample will be diluted twice and then loaded for four replicatesresulting in a total of eight wells of each of the six samples. Thisprotocol will be followed for three days. Selection of specimens will bedone such that some of them will be clearly negative, some will beclearly positive and some will be weakly positive or just near thecutoff of the assay. These results will then be used to calculate meanU/mL values, standard deviations, and percent CV.

Summary of EBV Assay Development

A multiplex assay will be developed using specific antigens as listedabove. Microsphere Luminex technology will be used for this assay.Microsphere or beads will be coated with specific antigens as listedabove and then optimized and validated in singlet and multiplexes.Optimization and validation will be performed using banked human subjectspecimens that will be diluted to note detection to establish cutoff andreference ranges.

Method of Treatment

In another aspect, the invention provides methods of treating the EBVsubset of ME/CFS patients. In some embodiments, the methods of treatinga patient with CFS involves evaluating the patient for serologicevidence of the presence of nucleic acid molecules that indicate primaryinfection by one or more CFS-causing agents, thereby detecting thepresence of each CFS-causing agent present in the patient; evaluatingthe patient for serologic evidence of one or more co-infection;determining whether one or more co-infections are present in thepatient; administering, or causing to be administered, to a patient atherapeutically effective amount of at least one pharmaceuticalcomposition; further comprising at least one antiviral agent such thateach CFS-causing agent found in the patient is effectively treated by atleast one antiviral agent administered to the patient; and, if one ormore co-infections are present, also administering, or causing to beadministered, to the patient therapeutically effective amount of atleast one pharmaceutical composition such that each co-infection foundin the patient is effectively treated by at least one pharmaceuticalcomposition administered to the patient, thereby treating the CFS. Eachpharmaceutical composition can comprise one active agent, or it cancomprise a cocktail of more than one active agent. The co-infection canbe with, for example, Borrelia burgdorferi, Streptococcus pyogenes,Ehrlichia chaffeensis, Babesia microti and Mycoplasma pneumonia. Theantiviral agent can be, for example, valacyclovir, valganciclovir,maribavir, famciclovir and foscarnet. However, any antiviral agent thatis effective against a CFS-inducing infection can be used according tothe methods disclosed herein.

Group A comprises CFS patients with EBV, HCMV, and/or HHV6 persistentinfection in single virus or combination, but without additionalco-infections. The following are the criteria for selecting thesepatients;

1) Patients meet international and CDC criteria for CFS and haveabnormal 24 hour ECG monitors (as determined by the presence oftachycardia and/or abnormal T waves). 2) Patients are positive for HCMV,EBV and/or HHV6, as determined by detection of serum antibodies to eachof these virus's or to gene products of these virus's, weather detectedusing enzyme-linked immunosorbent assay (ELISAN) or other methods,including those of the invention. 3) Patients are negative for CFSco-infections, such as for example Borrelia burgdorferi, Streptococcuspyogenes, Ehrlichia chaffeensis, Babesia microti and Mycoplasmapneumonia. These patient respond favorably, there are validated energyindex point scores increase from less than to greater than 6, usuallyreaching an energy point score (EI) of 7-9 within six to twelve monthsantiviral therapy with appropriate antiviral agents, including, forexample valacyclovir, valganciclovir, maribavir, famciclovir andfoscarnet.

Group B comprises CFS patients with EBV, HCMV and/or HHV6 persistentinfection, either alone or in combination, but also having one or moreco-infections. The following are the criteria for group B patients;

1) Patients meet international and CDC criteria for CFS and haveabnormal 24 hour ECG monitors (as determined by the presence oftachycardia and/or abnormal T waves). 2) Patients are positive for HCMV,EBV and/or HHV6 infection. 3) Patients are positive for one or more ofthe following: Borrelia burgdorferi, Streptococcus pyogenes, Ehrlichiachaffeensis, Babesia microti and Mycoplasma pneumonia.

CFS patients in group B can be treated with for example, valacyclovir,valganciclovir, maribavir, or other derivatives or benzimidazole, asappropriate to the particular infections present in a particularpatient. famciclovir and foscarnet. CFS patients in group B can also betreated with appropriate therapy for co-infection, which may include,for example infection with Borrelia burgdorferi, Streptococcus pyogenes,Ehrlichia chaffeensis, Babesia microti and Mycoplasma pneumonia.Generally, unless group B patients are treated for the co-infection theywill not improve.

Infection with Borrelia burgdorferi can be diagnosed by, for example,detecting the presence of IgM or IgG to Borrelia burgdorferi usingWestern blot or ELISA. It may be possible that IgG is not detected in apatient with CFS, in which case a positive IgM result would bediagnostic of infection with Borrelia burgdorferi. Antigens used forthis assay are exact prototypes used by US Centers for Disease Control.

Infection with Borrelia burgdorferi can be treated with, for example,intravenous (IV) ceftriaxone 0.1-5 gm, for example 1.0-1.5 gm,intravenous piggy-back (IVPB) every 12 hours for 30 days, followed byoral amoxicillin 0.01-4.0 gm, for example 0.5-0.75 gm, 4 times/day untilabove serum tests negative. IV penicillin G or its equivalent every 6-8hours can substitute for ceftriaxone. For treatment of Borreliaburgdorferi, as well as of any other secondary infection describedherein, qualified health care personnel can prescribe appropriatedosages for effective treatment of CFS. Any dosage that falls within thescope of sound medical judgment is contemplated as part of thisinvention.

Adult rheumatic fever is caused by a hyperimmune response to, forexample, Streptococcus pyogenes infection. It can be diagnosed by, forexample, finding an elevated antistreptolysin 0 (ASO) titer (LabCorp,Dublin, Ohio); for example, an ASO titer over 400 units would bediagnostic of adult rheumatic fever or co-infection by Streptcoccuspyogenes. Adult rheumatic fever may also be accompanied by, for example,thickening of the aortic and/or mitral valve, which can be viewed on anechocardiogram.

Adult rheumatic fever can be treated with, for example, ceftriaxone orpenicillin G as above followed by bicillin 0.1-5 Mu, for example 1.2 Mu,every 2-4 weeks until ASO titer is less than 200. A CatScan ofsinuses/mastoids may be indicated to exclude obstructive sinusitis.Bicillin may be necessary for 2-4 years.

Babesiosis can be diagnosed by, for example, finding an elevated serumtiter for IgG to Babesia microti. Chronic Babesiosis can be treatedwith, for example, Ataquavone 100-1500, for example 750 mg orally, plusazithromycin 0.01-4 gm, for example 0.5 gm, twice daily for 6 weeks.

Detection of IgM to Babesia Microti can also be done.

Ehrlichia chaffeensis co-infection can be determined by, for example,detecting a positive serum titer for IgG or IgM to Ehrlichiachaffeensis. Chronic Ehrlichiosis can be treated with, for example, IVdoxycycline 10-1000 mg, for example 100 mg, every 12 hours for 4, 6, or12 weeks.

Infection by Mycoplasma pneumoniae can be diagnosed by, for example,finding a markedly positive serum titer for IgG or IgM to Mycoplasmapneumoniae. Infection by Mycoplasma pneumoniae may also be accompaniedby, for example, an abnormal standard 12-lead electrocardiogram.

Mycoplasma pneumoniae myocarditis can be treated with, for example, IVdoxycycline plus/minus IV azithromycin for 6 weeks.

Effective Sublassifications of CFS Patients have not been PreviouslyRecognized.

Pharmaceutical Compositions and Administration

In general, to provide a therapeutically effective amount of theantiviral agent, a suitable effective dose will be in the range of 0.1to 20 grams a day and preferably in the range between 0.3 to 15 gramsper day, more preferably about 0.5 to 10 grams per day. The dosage ofcourse, varies with the body weight of the patient up to a 70 kgindividual, a dose of 4 grams per day may be appropriate (e.g., 10 mgper KG valacyclovir every six hours). The desired dose can be presentedas two—four or more smaller doses administered at appropriate intervalsthroughout the day. These smaller doses may be administered in unit'sdosage forms. For example, for valacyclovir and famciclovir, the dosagecan be, for example 14 mg/kg every 6 hours (1.0 g every 6 hours for a 70kg person). The dosage of valacyclovir and famciclovir can be, forexample, up to or at least about 0.5-8 grams every 6 hours. The dosageof valgancyclovir can be, for example, from about 450-900 mg every 12hours, or up to or at least about 100-2000 mg or more every 12 hours,depending on for example patient weight and tolerance. For maribavir thedosage can be, for example, for about 400-500 mg every 8 hours or forexample up to or at least about 100-1000 or more every 8 hours.Qualified health care personnel can prescribe appropriate dosages foreffective treatment of CFS. Any dosage that falls within the scope ofsound medical judgment is contemplated as part of this invention.

In particular for valacyclovir, or a derivative such valacyclovirhydrochloride, a patient can be administered a dosage in the range of0.1 to 50 mg/kg of body weight of the patient per dosing interval,generally every 6 hours. The dosing interval is determined by thebioavailability of the antiviral agent and its excretion from the body.For example, the patient can be administered a dosage in the range of0.3 to 40 mg/kg of body weight of valacyclovir hydrochloride orallyevery 6 hour. For example, a patient can be administered 10 mg/kg ofbody weight valacyclovir hydrochloride every 6 hours.

The treatment period for a CFS patient varies on a case-by-case basis.It is believed that for some, CFS is an ongoing and persistent problemrequiring continued treatment. The duration of the therapy depends onthe intensity of the CFS as affected by the therapy. One indicator of animprovement in EBV-isolated CFS patients is a decrease of a level of IgMantibodies to viral capsid antibodies (VCA) for EBV. Generally, thetherapy duration is proportional to the intensity of the CFSmanifestation. Accordingly, following administration of an antiviralagent, supplemental tests are helpful to check for recurrent CFS and todetermine the treatment duration. The duration of treatment may be 6-18months or longer or shorter as determined by the attending physicianusing the methods described herein.

Antiviral agents which demonstrate anti-herpetic action, such as thosespecific to, for example, EBV, HCMV, or HHV6, can be used for thetreatment of chronic fatigue syndrome. Such antiviral agents may beeffectively administered, for example, by oral methods, or as largerdoses in time delay formulations. Included among this group of antiviralagents are valacyclovir, valganciclovir, maribavir, famciclovir andfoscarnet and other herpetic antiviral agents and pharmaceuticallyacceptable derivatives of these antiviral agents. Such pharmaceuticallyacceptable derivatives include salts, hydrolysable esters and chelatesof the antiviral agents and such similar derivatives which have nonegative pharmaceutical effect on the patient upon administration andare thus “pharmaceutically acceptable”. A pharmaceutically acceptablesalt can become a for example, an acidic salt derived from anappropriate acid, for example hydrochloric, sulfuric, phosphoric,maleic, fumaric, citric, lactic, tartaric, acetic or p-toluenesulphonicacid.

While embodiments of the invention have been illustrated and described,it is not intended that these embodiments illustrate and describe allpossible forms of the invention. Rather, the words used in thespecification are words of description rather than limitation, and it isunderstood that various changes may be made without departing from thespirit and scope of the invention.

What is claimed is:
 1. A method of diagnosing an Epstein-Barr virusbased Myalgic Encephalomyelitis-Chronic Fatigue Syndrome patient,comprising the step of: Identifying Epstein-Barr virus Abortive Lyticreplication by determining the presence of serum antibodies to EBVencoded dUTPase or EBV encoded DNA Polymerase in the patient.
 2. Themethod of claim 1, wherein the step of determining the presence of EBVencoded dUTPase and DNA Polymerase antibodies further comprises thesteps of: obtaining a blood sample from the patient; and analyzing theblood for the presence of serum antibodies to EBV dUTPase or EBV encodedDNA Polymerase.
 3. The method of claim 1, wherein the antibodies aredetected using a neutralization assay.
 4. The method of claim 1, whereinthe antibodies are detected using a multiplex immunoassay.
 5. The methodof claim 1, wherein the antibodies are detected using a LuminexMultiplex Immunoassay.
 6. A method of diagnosing an Epstein-Barr virussubset of Myalgic Encephalomyelitis-Chronic Fatigue Syndrome patients,comprising the steps of: i. Determining the presence of encoded EBVEarly Antigen, Diffuse; ii. Determining the presence of EBV encoded DNApolymerase; iii. Determining the presence of EBV encoded dUTPase; andiv. Diagnosing a patient with Epstein Barr Abortive Lytic Replicationwhen the presence of EBV Early Antigen, Diffuse is found in conjunctionwith the presence of EBV encoded DNA polymerase or EBV encoded dUTPase.7. The method of claim 6, wherein the step of determining the presenceof EBV encoded dUTPase further comprises obtaining assays to detect thepresence of serum antibodies to the early EBV non-structural proteins ofthe EBV tegument.
 8. A method of diagnosing the causation agent for aMyalgic Encephalomyelitis-Chronic Fatigue Syndrome patient, comprisingthe step of: a. Determining the absence of Epstein-Barr virus AbortiveLytic replication, comprising the steps of: i. Determining the absenceof serum antibodies to EBV VCA IgM; ii. Determining the absence of serumantibodies to EBV encoded DNA polymerase; and ii. Determining theabsence of serum antibodies to EBV encoded dUTPase.
 9. The method ofclaim 8, wherein the absence of EBV encoded dUTPase is furtherdetermined through assays for serum antibodies to the early EBVnon-structural proteins of the EBV tegument.
 10. A method of diagnosingand treating a subset of patients with Myalgic Encephalomyelitis-ChronicFatigue Syndrome, comprising the step of: Identifying Epstein-Barr virusAbortive Lytic replication in patients with MyalgicEncephalomyelitis-Chronic Fatigue Syndrome through the use of serumantibodies to EBV dUTPase and EBV DNA Polymerase as molecular markers;and treating the subset of patients with the administration of atherapeutically effective amount of at least one antiviral agent. 11.The method of claim 10, wherein the antiviral agent is selected from thegroup consisting of valacyclovir, valganciclovir, maribavir, famciclovirand foscarnet.
 12. The method of claim 10, wherein the antiviral agentis valacyclovir.
 13. The method of claim 10, wherein the antiviral agentis administered for a period of over 12 months.
 14. A method ofdiagnosing an Epstein-Barr virus subset of MyalgicEncephalomyelitis-Chronic Fatigue Syndrome patients, comprising thesteps of: a. Positive screening criteria for ME/CFS; b. Testingserologic levels for the following indicia: i. The absence of IgM, IgGBorrelia burgdorferi; ii. The absence of Babesia microti, IgM, IgG; iii.The absence of Ehrlichia phagocytophilia; iv. positive serum titer forIgG or IgM to Mycoplasma pneumonia; and v. Antistreptolysin 0 titerlevels at less than 400; c. Testing for abnormal cardiac functionality;and d. Detecting the presence of serum antibodies to EBV dUTPase or EBVDNA polymerase.